Gene, 13 (1988) 419-426

Elsevier

419

GEN 02195

Isolation of overproducing recombinant mammalian cell lines by a fast and simple selection procedure

(Recombinant DNA; gene transfer in mammalian cell lines; combined selection; methotrexate resistance; puromycin resistance; gene dosage effect; chromosomal localization; gene expression)

M. Wirth’, J. Bode”, G. Zettlmeisslb and H. Hauser”

a Cell Biology and Genetics Section, GBF, D-3300 Braunschweig (F.R. G.), and b Research Laboratories of Behringwerke AG. D-3550 Marburg (F.R. G.) Tel. (6421)392319

Received 17 March 1988 Revised 7 July 1988 Accepted 14 August 1988 Received by publisher 21 September 1988

SUMMARY

The expression of genes transfected into mammalian cells is critically dependent on both the copy number and the site of integration. This conclusion was derived from the transfection of a plasmid containing three linked genes, which guarantees the same dose of each gene in the recipient cell clones. The selection pressure imposed by the usual concentrations of the neomycin analogue G418 or puromycin alone is low and yields cell clones with widely differing transcription rates. Time-consuming screening procedures, which are generally applied to obtain high-yield cell lines, can be obviated by the simultaneous transfer of two drug-resistance genes (neo plus dhfr, or neo plus pat). By a combined selection procedure only cells with high-level expression for the first marker will survive upon exposure to increasing concentrations of the second antibiotic. Generally, these cells also exhibit high expression levels for the non-selected gene. Many of the selected clones exhibit increased copy numbers of the respective gene but some of them owe their high productivity to a favorable position of only one or a few copies of the gene. Screening for high-yield clones using the combined selection protocol introduced here provides rapid and simple access to authentic and mutant gene products.

Correspondence 10: Dr. H. Hauser, Gesellschat? fllr Biotechno- logische Forschung, Mascheroder Weg 1, D-3300 Braunschweig (F.R.G.) Tel. (531)6181235.

Abbreviations: Ap, ampicillin; ATIII, antithrombin III; ATCC, American Type Culture Collection; BHK, baby hamster kidney; bp, base pair(s); CAT, Cm acetyltransferase; cat, gene coding for CAT; CHO, Chinese hamster ovary; Cm, chloramphenicol; DHFR, diiydrofolate reductase; dhj?, gene coding for DHFR;

DMEM, Dulbecco’s modified Eagle’s medium; ELISA, enzyme-

linked immunosorbent assay; G418, geneticin; HSV, Herpes sim- plex virus; IFN, interferon; kb, kilobase or 1000 bp; I.&-, thymidine kinasedeficient mouse L cell; MTX, methotrexate; NCS, newborn calf serum; neo, gene coding for aminoglycoside 3’-phosphotransferase from transposon Tn5; pat, gene coding for puromycin N-acetyltransferase; R, resistance; SV40, simian virus 40; rk, thymidine-kinase-coding gene.

0378-I 119/88/$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)

420

I~RODU~ON

Beginning with experiments of Szybalska and Szybalski (1962), DNA-mediated gene transfer has widely been used to introduce foreign genes into animal cells. This technique enables one to define regions within or around certain genes that are required for their regulation, or to produce authentic proteins of academic, pharmaceutical or industrial interest in large quantities. Furthermore, the effect of mutations on functional or structural properties of a protein can be tested readily by expression of the mutated gene in such cells.

Various methods for gene transfer into animal cells have been described (i.e., Graham and Van der Eb, 1973; Neumann et al., 1982; Schalfner, 1980; Cepko et al., 1984). Depending on the technique used, the transferred gene(s) can integrate as a single copy or multiple copies. The level of expression is determined by many factors, such as copy number, locus of inte~ation and the strength of both pro- moter and enhancer. Moreover, correct processing and stability of the transcribed RNA as well as sta- bility and non-toxicity of the gene products are of importance. Among the factors determining produc- tion, the copy number can be manipulated whereas efficient methods for targeting genes to loci of high transcriptional activity are still lacking. A system of screening for high-yield clones by immunological or biological assays has therefore generally proven to be very time-consuming. As the level of expression can vary with a given gene, efficient expression systems become more and more interesting.

Because there is some relation between expression levels and the gene’s copy number, systems providing the possibility for amplification are of interest. In the last few years several mammalian genes amplifiable under selection pressure have been identified (Stark, 1984). Among these, the DHFR-coding gene (&jr) which is amplitied in response to increasing concen- trations of the enzyme’s inhibitor, MTX, has been well characterized. Kaufman and coworkers have shown that a CHO &$- cell line is well suited to detect ~p~cation of exogenous dhf and linked genes (Kaufman and Sharp, 1982a). However, screening for high product yield is time-consuming. So far, MTX screening of dhf amplification is rather limited to the CHO system, because transfer to other, non-&j? lines is frequently accompanied by

non-specific resistance, and because competition with the endogenous d&? gene cannot be excluded.

We have developed a method for direct screening of high-yield wild-type cell lines following a simul- taneous cotransfer of two selectable genes (the neo gene plus either a dhf gene or the pat gene). The combined selection procedure uses G4 18 plus MTX or G418 plus puromycin and increasing concentra- tions of the second agent (MTX or puromycin, respectively). Owing to the use of two selectable markers, this system avoids the d~culties accom- panied with nonspecific resistances.

MATERIALS AND METHODS

(a) Plasmids

The construction of the 10. I-kb plasmid pCATIFfi (see Fig. 1) horn sequences coding for human IFN-8, bacterial CAT, and bacterial n~rnyc~ resistance has been described (Klehr and Bode, 1988). The IFN-fi gene is under the control of its endogenous promoter, the neo is fused to the constitutive pro- moter of HSV and cat is transcribed from the SV40 early promoter. The plasmids pSVATII1 and pSVtss + , harboring the cDNA genes of AT111 and IFN# (HincII-fragment) under the control of the SV40 early promoter were previously described (Zettlmeissl et al,, 1987; Reiser and Hauser, 1987). Plasmid resistance markers used are: pAG60 (Colb~re-Graph et al., 1981), pAd&j? ( = pA- dD26SV(A)-3) (Kaufman and Sharp, 1982a) and pSV2pac (Vara et al., 1986). pSVZpac codes for puromycin N-acetyltransferase from Streptomyces

alboniger providing a new dominant selection system (Vara et al., 1986). Expression of the plasmid confers resistance to puromycin.

(b) Cell culture and DNA transformation

Ltk- (Kit et al., 1963), BHK-21 (ATCC CCL-IO) and CHO DUKX Bl (Urlaub and Chasin, 1980) cells were maintained in DMEM supplemented with 10% NCS, antibiotics and for CHO cells with thymidine, adenosine and deoxyadenosine accord- ing to Urlaub and Chasin (1980). Plasmids were transfected as linearized or as supercoiled circular DNA as stated, using the Ca * phosphate precipi-

421

tation technique with the indicated amount of DNA (Graham and Van der Eb, 1973). The medium was changed 4 h prior to transfection. The precipitates in a final volume of 0.5 ml were added to 5 ml medium over 3 x lo5 cells in 25 cm2 flasks. After incubation for 16 h, the medium was renewed. Selection with the indicated amounts of MTX, puromycin and/or G418 in DMEM/NCS was started 24 h later. At this time BHK and CHO cells were split 1: 3 if selected only with G418. Clone numbers were determined by microscopic counting of colonies after six to twelve days. Single clones were obtained by replating the cells in microtiter plates using the limited-dilution method.

(c) Determination of chloramphenicol acetyltrans-

ferase activity and human interferon secretion

CAT assays were performed essentially as de- scribed by Scholer and Gruss (1984). Extracts were prepared from 2 x lo6 cells grown to confluency, counted and assayed for CAT enzyme activity. CAT activity is given in pmol of acetylated Cm per min of incubation and per 1 x lo6 cells. Acetyl CoA was purchased from Pharmacia and [ 14C]Cm (spec. act. 50-60 mCi/mmol) was obtained from Amersham. Human IFN secretion was determined by an anti- viral assay (Finter, 1969) in supernatants collected 15 h after treatment of confluent cells with poly(1) : poly(C) for 5 h (Dinter and Hauser, 1987). CAT enzyme activity and interferon secretion were later normalized to the amount of cells used for the respective assay. Due to the dilutions chosen, inter- feron titers are precise to a factor of 2. For simplicity, the results have been grouped in four major classes, both for IFN titers and CAT activities.

and dot-blot analysis have been described (Bode et al., 1986). For further evaluation, cell clones were categorized in four groups according to copy number (see Fig. 2). Since low copy numbers were predomi- nant, separate groups could be formed for clones containing a single copy or two copies, respectively. Accordingly, larger ranges had to be chosen for the remaining multicopy clones.

RESULTS AND DISCUSSION

(a) Gene copy number per cell versus expression levels

(d) Determination of antithrombin III secretion

b

To evaluate the primary parameters determining the expression of transfected genes, we have con- structed the plasmid depicted in Fig. 1. The construct contains three linked genes coding for neomycin resistance (used as a selective marker), for the in- ducible ZZW-#I gene and the constitutive cat gene. After linearization, using the unique iWe1 site (which

a

Cells (5 x 105) were seeded in a 25-cm2 culture flask. After 24 h fresh medium lacking serum was added for an additional 24 h. The cells were counted and AT111 in the culture medium was measured by ELISA, as described earlier (Zetthneissl, 1987).

(e) Analysis of cellular DNA

Purification of genomic DNA from BHK and Ltk- cells and its characterization by Southern blot

N E E E N

0.8 2.8 2.5

Fig. 1. Structure of plasmid pCATIF/?. prior to transfection, the

plasmid (a) was linearized at its unique Me1 site (N) and puri-

fied. (b) The transfected state for a single copy is depicted below

the construct. E indicates the EcoRI cleavage sites. Fragment

sizes as specified in kb. Symbols: n , cos sequence from phage rl;

0, pBR origin of replication.

422

increased the number of clones two-fold), the plas- mid was transfected into Lfk- cells and clones were selected by their resistance to G418. Carrier DNA was omitted since it was reasoned that such an approach would maximize the influence of the sites of ~t~ation into the host’s genome.

Among the 600 cell clones obtained durhig au optimized transfection protocol, 85 were probed for IFN-p synthesis. Of these 90% produced amounts detectable by the bioassay (i.e., more than 5 units per lo6 cells) and 40% more than 100 units. Only the latter ~$oup (36 clones) was subjected to further study (Fig. 2).

For a characterization of the chromosomal status, genomic DNA from the 36 clones was isolated, cut by EcoRI or EcoRI + NdeI, blotted to a nylon membrane after el~~ophoresis and probed with the

COPIES/CELL

1 2 3.30 , 3t-100

C :LONES/GROUP

COPIES/CELL

-

CLONES/GROUP

Fig. 2. Relation between the number of integrated copies and gene expression in Lzk- cells. I.&- cells were transfected with 10 fig of linearized pCATIFg (Fig. I) and selected (7OO~~ml G418) as described in MATERIALS AND METIIODS. Isolat- ed cell clones were grown and tested for IFN-/I secretion upon induction, for their steady-state levels of CAT enzyme activity and their content of heterologous DNA. (Panel a) Levels of IFN production by 36 clones producing 100 units per lo6 cells or more (length of solid bars). Clones have been divided into four groups along the abscissa according to their copy number. The width of each bar is proportional to the number of clones in each group. The number of gene copies per cell, averaged over the entire popufation, was 7. (Panel b) Levels of CAT activity in extracts from the same clones analysed above. Symbols are the same as in panel a.

nick-translated plasmid. Copy numbers were deter- mined from the hybridization intensities of the 2.8kb and 4-kb EcoRI fragments which were intact in all cases (cf., Fig. 1). At low copy numbers, the 3.3-kb EcoRI fragment was lost by integration and replaced by distinct high-& fruits ~clu~ng host DNA. At copy numbers higher than ten, variable amounts of the 3.3-kb fragment were reformed as expected for an ordered tandem array of integrated DNA seg- ments. During integration, the N&I sites were lost in all cases suggesting minor modifications at the ends of the DNA molecules (not shown).

Clones were divided into four groups according to their copy number and tested for their capacity to produce IFN-8 and CAT (Fig. 2). In most clones having integrated more than three copies, the levels of IFN-/? and CAT production ran parallel.

The results compiled in Fig. 2 clearly show that gene activity can vary considerably. Within the detectable range, levels of IFN-band CAT vary 400- and loo-fold, respectively. A high copy number may be favorable for high yields but this is clearly not generally the case. The largest variation of activities is evidently observed in the range of one to ten copies per cell. In a number of cases, this class yields a per-cell production which is not reached by multi- copy integrates.

Considering the fact that in most clones the activity of the linked genes is parallel, very low levels of the raeo gene product s&ice for survival. Thus selection for maximum levels of expression is not achieved by using a single selective marker.

(b) Selection among baby hamster kidney cells with various drug combinations

Several authors showed that cotransfection is an efficient way for s~~t~~usly ~~~uc~g non- linked genes into animal cells (Wigler et al., 1979; Huttner et al., 1979; Wold et al., 1979; Hsiung et al., 1980; Kaufman and Sharp, 19825). Usually, co- transferred DNA fragments become integrated in direct proximity to each other and in the majority of cases they are coordinately expressed. We have selected cotransfectants (derived from BHK and Ltk- cells) of a selective DNA (&p) and an expres- sion plasmid with increasing amounts of a selective drug (MTX), Although expression roughly corre- lated with drug concen~ation, a detailed analysis of

the resulting cell clones showed that unspecific drug resistance occurred frequently (not shown). Since the cotransfection procedure is not limited to the number of genes to be introduced, the procedure can be extended to more than two genes. Before entering any time-consuming screening protocols, we have investigated the effect of simultaneous selection with G418 and MTX. pSVATII1, the expression plasmid for human ATIII, was cotransfected with selection plasmids containing the neo gene (pAG60) and the dhf cDNA (pAti&) into BHK cells. G4 18 alone or G418 and increasing amounts of MTX were added 48 h post transfection to the culture medium. A dramatic cell lolling, increasing with high MTX con- centrations, was observed two to three days later. Stable transformants arose six to ten days post selec- tion. Fig. 3A shows that the number of clones per transfection declined rapidly with increasing amounts of MTX, while the amount of AT111 secreted reached a four-fold higher level than with single selection. Expression levels could be enhanced further with 600 nM MTX, in which case the initial number of cells transfected had to be enlarged (not shown). To evaluate the molecular basis of this phenomenon, we prepared high-M, DNA from respective transfectants and determined ATIII- specific DNA by hybridization using 32P-labeled AT111 cDNA. A typical dot blot analysis is inserted in Fig. 3A, providing evidence that increased expres- sion parallels DNA copy numbers (32-fold at 600 nM selection). The results clearly demonstrate that the resistance to increasing MTX concentra- tions roughly correlates with the mean copy number in the clone mixtures and with the expression of ATIII. This enables one to screen for high-produc- tivity recombinant BHK cell lines by using a com- bined G418/MTX selection procedure. A very simi- lar combined transfection/selection protocol for AT111 expression was recently described by Zetthneissl et al. (1988), where it was shown that after primary combined G418/MTX selection the average gene copy numbers of all transfected genes (A TZZZ, dhj'r and neo) and the AT111 synthesis levels could be further increased by secondary selection in stepwise increasing concentrations of MTX (up to 10 PM).

We examined whether the combined selection procedure to obtain high-expression recombinants is limited to the G418/MTX system or is also applica-

0

t

! 423

5 = N \

4 ! al lJ

3 a 0

7

2 Y

= 1 -

t

I I I 1 I I I

0 100 200 300 coo 500 600

Methotrexote ( nM 1

In t t

F -5 z4 \

ul -4 =

0, u

-3 “0

7

-2 Y

=

-1 r =l

t I I 1 I I 1 I

0 5 10 15 20 25 30

Puromycin (pg/ml)

Fig. 3. AT111 production and transformation efficiency upon combined selection of BHK cells. (Panel A) BHK cells (1.2 x 10’) were transfected with 0.8 Kg of pAddhfr, 0.2 pg of pAG60 and 2pg of pSVAT’II1. Selective medium contained 500 pg G418/ml and the indicated amounts of MTX. AIter selec- tion, the resistant clones were counted, propagated as a mixture of clones and seeded for ATIII determination. Chromosomal DNA from aliquots of the cultures was prepared, denatured and dotted in serial 1: 1 dilutions on nitrocelhdose paper. The dot blots were hybridized to 32P-labeled cDNA fragment from pSVATII1 and autoradiographed. The dot lanes corresponding to cells selected at 0, 100 and 600 nM MTX are shown in the insert. Symbols: C, DNA from control cells. Squares, number of stable transformants; circles, amount of secreted ATIII. (Panel B) BHK cells were treated as described in panel A, except that a puromycin resistance gene/selection-system was used instead of the dhj? gene/MTX-system. Plasmid pSV2pac (0.8 rg) replaced PAD&? at the transfection. For selection, the indicated concentrations of puromycin were used in combination with G418 (500 &ml). (Symbols as in panel A.)

424

ble to the comb~ation of other selection markers. pSVATII1 was cotransfected with pAG60 as the basal selection marker and pSV2pac (as the marker on which selection screening is applied). Stable transformants were achieved by simultaneous selec- tion with G418 and increasing concentrations of puromycin. As in the G418/MTX system the num- ber of clones per transfection declined with increas- ing concentrations of puromycin, i.e., this marker combination is also capable of screening for high- secretion r~ornb~~t cells as demons~ated in Fig. 3B. The amount of AT111 secreted into the superuatant of BHK cells increased by a factor of 4 and 6, if the selection was carried out with 7.5 pg or 30 pg puromycin per ml as compared to the basal selection with G418 alone. Very similar results were also obtained using the combination of pSV2puc and pAddhfr as selectable constructs with puromycin and increasing amounts of MTX as selecting agents (not shown).

(c) Appli~tion of the combined selection procedure to other cell systems

The ability to use combined selection for the enhanced synthesis of AT111 in BHK cells en- couraged us to examine whether this screening method is also applicable to other mammalian cell lines. CHO and mouse L cells are widely used for transfection experiments and especially for produc- tion of glycoproteins. Therefore, we introduced plas- mids pSVtss+ together with pAG60 and pSV2pac into mouse L and CHO cells. pSVtss + harbors the structural gene for IFN-fi. The amount of IFN-/I secreted by the pool of recombinant cells was deter- mined. The results in Fig. 4,A and B indicate that a combined selection screening with G4 18/puromycin is applicable to both mouse L cells and CHO cells. The enhancement of IFN-jl synthesis obtained by combined selection of CHO cells was unexpectedly high. Clones selected at 10 pg/ml of puromycin secreted 20-fold more IFN-/I than cells selected with G418 alone. Compared with the conventional way to obtain CHO cells producing comparable levels of IFN-@ (screening of single cell clones and selection for amplification, cf., Reiser and Hauser, 1987) the combined selection protocol described here is straightforward.

We would like to note that in principle the direct

selection for ~~-pr~ucti~ty cell clones does only require one selection system. A second selection marker, however, greatly reduces the probability of obtaining spontaneous drug resistance. Indeed, in a direct screening with a very high concentration of the selectable drug a second selection is not necessary. In this case, the transfection conditions must be optimized to obtain resistant clones.

:, 10000 ;

i - t I

5 10

Puromycln ( pg/ml 1

5 10

Puromycbn Cpg/ml)

Fig. 4. Combined selection in Lrk- and CHO cells. The experi- men&I outfines are as described in Fig. 3. CHO and U/c- ceils were transfected with 2 ~g pSVtss + ,0.2 pg of pAG60 and 0.8 yg of ~SV~JWC. AtIer selection with 700 ug G418jml for Lfk- cells, 360 pg C%tS/ml for CHO cells in combination with the indicated amounts of puromycin the number of stable transformants was counted and the human IFN-fi secretion was determined. Com- bined selection with concentrations of 30 and 100 pg/ml of puro- mycin revealed no stable transformants. (A) IN’ cells; (B) CHO cells. Squares, number of stable transformants; circles, IFN-/I secretion,

42s

Zettlmeissl et al. (1987) showed that MTX amplification of transfected dhfr and adjacent AZ711

DNA in CHO cells results in higher expression levels when using a promoterless and therefore weakly expressed dhfr cDNA. Similar data were obtained for a promoterless bk gene and respective selection of Ltk- cells (Roberts and Axel, 1982; Israel et al., 1987). We anticipate that the comb~ation of this technique with the combined selection procedure reduces the amount of the respective drugs and could even result in a higher extent of the screening capacity.

The dot blot analysis shown in Fig. 3A indicated that expression level of the selected cell clones corre- lates with the mean number of integrated expression plasmids. On the other hand, the results obtained in the experiment illustrated in Fig. 1 suggest that high- yield clones may contain only very few copies of transfected DNA. Therefore it was of interest to investigate whether such cells were part of the clone mixture obtained with the combined selection proce- dure. After subcloning the best producing cell clones were subjected to hybridization analysis to define the copy number of transferred expression plasmids. In Fig. 5, DNA from these cell clones was dot-blotted and probed with IFN-fi DNA. The data clearly de- monstrate that high-productivity subclones with low copy number of transfected DNA are present in the

total population. Therefore, with this procedure

high-yield cell clones are obtained irrespective of whether this is caused by gene dosage or by favorable sites of ~t~ation.

(d) Conclusions

(i) Our results demonstrate that high-level expres- sion of recomb~~t cell lines is largely determined by both the copy number of transfected DNA and its integration locus.

(ii) Coexpression of two selection markers to- gether with the gene of interest and screening with a combined selection afterwards facilitates the iso- lation of cells with a high expression level.

(iii) The combined selection procedure is simple and fast. The procedure works well with several selective markers in combination with nonselected genes in various cell types.

(iv) Cells obtained by the combined selection contain clones, in which high-level production is cor- related with bigb copy number of the integrated ex- pression plasmid, as well as clones harboring the plasmid in favorable integration sites within the c~omosom~ DNA.

Thus, the ease and speed of combined selection together with the high-level expression achieved, makes the procedure of particular interest for functional or structural analysis of mutant glyco- proteins by overexpression of the altered gene in animal cells.

CLONE B 9

* a * * * e I) CLoNEF’l

@ * CLONE X tV

Fig. 5. Compa~son of the DNA content of in~~du~ cell clones obtained by combined selection. In analogy to the experiments described in Fig. 3A, BHK cells were subjected to combined selection after cotransfection with pSVtss + , pSV&ihfi and pAG60. At 100 nM MTX a mixture of 460 cell clones producing lo5 units of IFN-/l per lo6 cells in 24 h was obtained. From this mixture, single-cell clones were isolated and screened for IFN production. The chromosome DNA of clones, al1 producing between lo5 and 5 x IO5 units of IFN per lo6 cells in 24 h was probed by dot blots (compare Fig. 3A) with a IFN-#J cDNA fragment from pSVtss + . ‘Standard’ represents a DNA dosage marker (pSVtss + ) and corresponds to 100 gene copies per cell.

We wish to thank K. Maass, S, Heiser, M. Ausmeier and C. Morelle for expert technical assistance, Dr. Jimenez (Madrid) for the plasmid pSV2pac, I. Do~und for typing and Dr. J. Collins for critical review of the manuscript.

REFERENCES

Bode, J., Pucher, H.-J. and Maass, K.: Chromatic s~cture and induction-dependent conformational changes of human inter- feron-&genes in amouse host cell. Eur. J. Biochem. 158 (1986) 393-401.

4.26

~pko,C.L.,Ro~~s,B.~~dM~~,R.C.:~ns~~tion~d applications of a highly transmissible mu&e retrovirus shuttle vector, Cell 37 (1984) 1053-1062.

Colbere-Garapin, F., Horodniceanu, E, Kourilsky, P. and Garapin, A.-C.: A new dominant hybrid selective marker for higher eukaryotic cells. J. Mol. Mol. 150 (1981) 1-14.

Dinter, H. and Hauser, H.: Superinduction of the human lnter- feron-/I promoter. EMBO J. 6 (1987) 599-604

Finter, N.B.: Dye uptake methods for assessing vual cytopatho- genie&y and their app~cation to interferon assays. J. Gen. Viii. 5 (1969) 419-427.

Gorman, C.M., Moffat, L.F. and Howard, B.H.: Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2 (1982) 1044-1051.

Graham, F.L. and Van der Eb, A.J.: A new technique for the assay of infectivity of human adenovirus 5 DNA. Viiology 52 (1973) 456-467.

Hauser, H., Gross, G., Bruns, W., Hochkeppel, H.-K., Mayr, U. and Collins, J.: Inducibility of human &interferon gene in mouse L-cell clones. Nature 297 (1982) 650-654.

Hsiung, N., Warrick, H., de Riel, J.K., Tuan, D,, Forget, B.G., Skoultchi, A. and Kucherlapati, R.: Cotransfer of circular and linear prokaryotic and eukaryotic DNA sequences into mouse cells. Proc. Natl. Acad. Sci. USA 77 (1980) 4852-4856.

Huttner, K.M., Scangos, G. and Ruddle, F.H.: DNA-mediated gene transfer of a circular plasmid into murine cells. Proc. Natl. Acad. Sci. USA 76 (1979) 5820-5824.

Israel, N., Chenciner, N. and Streeck, R.E.: Amplifiable expres- sion vectors for mammalian cell lines. Gene 51 (1987) 197-204.

Kaufman, R.J. and Sharp, P.A.: Construction of a modular dihydrofolate reductase cDNA gene: analysis of signals utilized for efficient expression. Mol. Cell. Biol. 2 (1982a) 1304-1319.

Kaufman, RJ. and Sharp, P.A.: ~plification and expression of sequences cotransfected with a modular dihydrofotate re- ductase complementary cDNA gene. J. Mol. Biol. 159 (1982b) 601-621.

Kit, S., Dubbs, D.R., Piekarski, L.J. and Hsu, T.C.: Deletion of thymidine kinase activity from L cells resistant to bromo- deoxy-urldine. Exp. Cell Res. 3 1 (1963) 297-312.

Klehr, D. and Bode, J.: Comparative evaluation of bovine papil- loma virus (BPV), vectors for the study of gene expression in mammalian cells. Mol. Gen. (Life Sci. Adv.) 7 (1988) 47-52.

Lindenmaier, W,, Hauser, H., Greiser de Wilke, I. and Schtltz,

G.: Gene shuttling: moving of cloned cDNA into and out of eukaryotic cells. Nucleic Acids Res. 10 (1982) 1243-1256.

Neumaon, E., Schaefer-Ridder, M., Wang, Y. and Hofsc~eider, P.H.: Gene transfer into mouse lyoma cells by electroporation ln high electric fields. EMBO J. 1 (1982) 841-845.

Reiser, W. and Hauser, H.: Recombinant human interferon-b from mammalian cell lines. Drug Res. 37 (1987) 482-485.

Roberts, J.M. and Axel, R.: Gene amplification and gene cor- rection in somatic cells. Cell 29 (1982) 109-119.

SchatBrer, W.: Direct transfer of cloned genes from bacteria to mammalian cells. Proc. Natl. Acad. Sci. USA 77 (1980) 2163-2167.

Scholer, H.R. and Gruss, P.: Specific interaction between e~~cer-cont~ng molecules and cellular components. Cell 36 (1984) 403-411.

Spandidos, D.A. and Wilkie, N.M.: Malignant ~~sfo~ation of early passage rodent cells by a single mutated human onco- gene. Nature 3 10 (1984) 469-475.

Stark, G.R. and Wahl, G.M.: Gene amplification. AMU, Rev. Biochem. 53 (1984) 447-491.

Szybalska, E.H. and Szybalski, W.: Genetics of human cell lines, IV. DNA-mediated heritable tr~sfo~ation of a biochemical

trait. Proc. Natl. Acad. Sci. USA 48 (1962) 2026-2034. Urlaub, G. and Chasm, LA.: Isolation of Chinese hamster cell

mutants deficient in dihydrofolate reductase activity. Proc.

Natl. Acad. Sci. USA 77 (1980) 4216-4220. Vara, J.A., Portela, A,, Or&, 3. and Jimenez, A.: Expression in

mammalian cells of a gene from ~~e~ro~~es ulboniger con- ferring puromycin resistance. Nucleic Acids Res. 14 (1986) 4617-4624.

Wigler, M., Sweet, R., Sim, G.K., Wold, B., Pellicer, A., Lacy, E., Maniatis, T., Silverstein, S. and Axel, R.: Transformation of mammalian cells with genes from procaryotes and eucaryotes. Cell 16 (1979) 777-785.

Wold, B., Wigler, M., Lacey, E., Maniatis, T., Silverstein, S. and Axel, R.: Introduction and expression of a rabbit &lobin gene in mouse fibroblasts. Proc. Natl. Acad. Sci. USA 76

(1979) 5684-5688.

Zettlmeissl, G., Ragg, H. and Karges, H.: Expression of biologi- cally active human ~ti~rornb~ III in Chinese hamster ovary cells. 3io~ec~olo~ 5 (1987) 720-725.

Zettlmeissl, G., Wirth, M., Hauser, H. and Ktlpper, H.: Efficient expression system for human antithrombmII1 in baby hamster kidney cells. Behring Inst. Res. Commun. 82 (1988) 26-34.

Communicated by H. van Ormondt.